Bioenergetic Profiling of Zebrafish Embryonic Development

Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility

Metalloprotease OMA1 Fine-tunes Mitochondrial Bioenergetic Function and Respiratory Supercomplex Stability

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism – intramitochondrial quality control (IMQC) – is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1- associated disease phenotypes in humans

Opa1 Is Required for Proper Mitochondrial Metabolism in Early Development

<div><p>Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. <i>OPA1</i> mutations in humans lead to autosomal dominant optic atrophy. OPA1 knockout mice lose viability around embryonic day 9 from unknown reasons, indicating that OPA1 is essential for embryonic development. Zebrafish are an attractive model for studying vertebrate development and have been used for many years to describe developmental events that are difficult or impractical to view in mammalian models. In this study, Opa1 was successfully depleted in zebrafish embryos using antisense morpholinos, which resulted in disrupted mitochondrial morphology. Phenotypically, these embryos exhibited abnormal blood circulation and heart defects, as well as small eyes and small pectoral fin buds. Additionally, startle response was reduced and locomotor activity was impaired. Furthermore, Opa1 depletion caused bioenergetic defects, without impairing mitochondrial efficiency. In response to mitochondrial dysfunction, a transient upregulation of the master regulator of mitochondrial biogenesis, <i>pgc1a</i>, was observed. These results not only reveal a new Opa1-associated phenotype in a vertebrate model system, but also further elucidates the absolute requirement of Opa1 for successful vertebrate development.</p> </div

Western blot analysis.

<p><b>A.</b> Representative Western blot for Opa1 yolk-less protein in MMC and Opa1 morphants. Opa1 protein is reduced at 24, 48, and 72 hpf. Differences were isoform specific. Samples from 24 hpf were imaged from a separate blot. Contrast was adjusted to improve visualization. *indicates isoforms selected for densitometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059218#pone-0059218-g001" target="_blank">Figure 1B</a>). Western blot results have been replicated with at least four independent injections <b>B.</b> Quantification of most intense Opa1 isoforms identified by Western blot. All values were first normalized to β-actin protein levels.</p

Phenotypic analyses of MMC (A, C, E, G, I, K, M, O) and TB (B, D, F, H, J, L, N, P) morphant embryos and larvae.

<p>Opa1 morphants at 24 hpf (<b>B</b>) have increased density or ‘graininess’ in the brain region (arrow) and smaller eyes. At 48 hpf, Opa1 morphants have hindbrain ventricle enlargements (arrow) and smaller eyes (<b>D</b>). Opa1 morphants at 48 hpf also have impaired circulation compared with MMC morphants and often has blood accumulation below the heart (arrow) (<b>F</b>). At 72 hpf, Opa1 morphants have larger yolk cells, smaller eyes, smaller hearts, small pectoral fin buds (<b>H</b>) and pericardial edema (<b>J</b>). Many Opa1 morphants had unlooped hearts (<b>L</b>). (<b>N</b>) is the same image as (<b>L</b>) with the heart margins outlined (solid line) and the midline indicated by a dashed line. By 96 hpf, the edema is still present and can involve the eyes (<b>P</b>).</p

Primers for RT-PCR.

<p>Primers for RT-PCR.</p

Eye area and heart rate analyses for MMC (black) and Opa1 morphants (grey).

<p><b>A.</b> Eye area was measured by tracing the circumference of individual eyes using AxioVision software. N = 9–12. *p-value <0.05, **p-value <0.01 by Student's 2-tailed t-test. <b>B.</b> Heart rates were measured by counting beats per min for individuals injected with MMC or TB morpholino. N = 34 (48 hpf), n = 44 (72 hpf). *p-value <0.01 by Student's 2-tailed t-test.</p

Gene expression changes in MMC morphants (black) and Opa1 morphants (grey) normalized to MMC morphant levels.

<p>Significant increases in gene expression <i>pgc1a</i> (a, p = 0.02) and <i>peo1</i> (b, p = 0.002) were observed in <i>opa1</i> morphants compared to MMC morphants. Error bars are shown +/- SEM, n = 5. P-values obtained by ANOVA.</p

Primers for XL-PCR for mtDNA deletion assay.

<p>Primers for XL-PCR for mtDNA deletion assay.</p

Antisense morpholino sequences.

<p>Antisense morpholino sequences.</p